A problem with hematopoietic stem cell (HSC) transplantation is the risk of primary or secondary graft failure. As an example, in patients with hematologic malignancies who underwent their first myeloablative allogeneic hematopoietic cell transplantation, primary graft failure is 5.5%. One year overall survival after re-transplantation due to primary graft failure without relapse is as low as 11%. Risk factors for primary graft failue are insufficient numbers of transplanted HSCs and the necessity to reduce the intensity of chemotherapy and radiation conditioning due to the poor physical condition of patients to be treated. Reduced intensity conditioning is also required for HSC transplantation in certain non-malignant diseases such as Fanconi anemia. We are about to initiate clinical trials on a combination therapy of rituximab and our recombinant, affinity- enhanced protein, Ad35K++, that depletes the complement inhibitory protein CD46 from the cell surface and thereby enhances in vivo antibody therapy in cancers. Unexpectedly, we have found that this protein may have a significant ex vivo use: Ad35K++ alone, when added to human CD34+ cells, dramatically enhances their ability to engraft in myelo-ablated animals. This has important implications for HSC transplantation. This project will move this HSC engraftment enhancement candidate towards clinical testing in humans. The public health implications of improving clinical outcomes in therapies that require engraftment are significant. Specific areas that would benefit from the results of the studies proposed here include: humanized mouse models that receive human HSCs, HSC gene therapy, and diseases that are treated by HSC transplantation. Among these are malignant diseases (leukemia, lymphoma, gliomas, neuroblastomas, and other solid tumors), myelodysplastic syndromes, genetic diseases, and inborn errors of metabolism. The specific aims of the proposal include: 1. To understand the mechanism of Ad35K++-mediated engraftment enhancement 2. To optimize the approach and achieve engraftment with lower HSC numbers 3. To test the engraftment enhancer in more challenging transplantation settings. In phase 2 we will extend the findings to non-human primates and begin translational activities leading to a pre-IND meeting to support clinical trials on this ex vivo enhancer. Since we have already begun cGMP manufacture of our lead candidate for combination cancer therapy with rituximab, clinical translation of the findings in this proposal should be straightforward. The commercial potential for this product is clear and there is a great benefit to HSC transplant patients if our early animal model results are verified in humans.